Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.     

Freezing at -800 degrees C distorts the DNA composition of bacterial communities in intestinal samples

Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.     

Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging

The N¹-acetylation of spermidine or spermine by spermidine/spermine N¹-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSAT deficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion. Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N¹, N¹¹-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polymine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes.     

Visceral obesity is associated with high levels of serum squalene

Objective: To investigate the impact of visceral obesity on cholesterol metabolism in normoglycemic offspring of patients with type 2 diabetes. Research Methods and Procedures: The proportion of intra‐abdominal fat (IAF) was measured by abdominal computer tomography, and serum cholesterol synthesis and absorption markers were determined by gas‐liquid chromatography in 109 normoglycemic offspring of patients with type 2 diabetes. Insulin action was measured with the hyperinsulinemic euglycemic clamp. The gene encoding squalene synthase (farnesyl‐diphosphate farnesyltransferase 1) was screened with the single‐strand conformation polymorphism analysis and direct sequencing. Results: Cholesterol synthesis markers correlated positively with IAF (r = 0.213 to 0.309, p ≤ 0.027) and negatively with the rates of insulin‐stimulated whole‐body glucose uptake (r = ?0.372 to ?0.248, p ≤ 0.010). However, serum squalene, the first measured precursor of cholesterol synthesis, showed a positive correlation with IAF (r = 0.309, p = 0.001) without any association with subcutaneous fat or insulin sensitivity. Variation in the farnesyl‐diphosphate farnesyltransferase 1 gene did not explain elevated serum squalene levels in viscerally obese subjects. From the cholesterol absorption markers, cholestanol was associated negatively with IAF and positively with whole‐body glucose uptake (p < 0.05). Discussion: High serum squalene levels are associated with visceral obesity but not with subcutaneous obesity. Whether this finding is causally connected to visceral obesity remains to be established.     

Effects of strength training on muscle strength characteristics, functional capabilities, and balance in middle-aged and older women

Progressive strength training can lead to substantial increases in maximal strength and mass of trained muscles, even in older women and men, but little information is available about the effects of strength training on functional capabilities and balance. Thus, the effects of 21 weeks of heavy resistance training--including lower loads performed with high movement velocities--twice a week on isometric maximal force (ISOmax) and force-time curve (force produced in 500 milliseconds, F0-500) and dynamic 1 repetition maximum (1RM) strength of the leg extensors, 10-m walking time (10WALK) and dynamic balance test (DYN.D) were investigated in 26 middle-aged (MI; 52.8 +/- 2.4 years) and 22 older women (O; 63.8 +/- 3.8 years). 1RM, ISOmax, and F0-500 increased significantly in MI by 28 +/- 10%, 20 +/- 19%, 31 +/- 34%, and in O by 27 +/- 8%, 20 +/- 16%, 18 +/- 45%, respectively. 10WALK (MI and O, p < 0.001) shortened and DYN.D improved (MI and O, p < 0.001). The present strength-training protocol led to large increases in maximal and explosive strength characteristics of leg extensors and in walking speed, as well to an improvement in the present dynamic balance test performance in both age groups. Although training-induced increase in explosive strength is an important factor for aging women, there are other factors that contribute to improvements in dynamic balance capacity. This study indicates that total body heavy resistance training, including explosive dynamic training, may be applied in rehabilitation or preventive exercise protocols in aging women to improve dynamic balance capabilities.     

Neuroprotection by taurine in ethanol-induced apoptosis in the developing cerebellum

Background

Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period.

Methods

The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC).

Results

Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls.

Conclusions

We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

     

Genetic influence on human lifespan and longevity

There is an intense search for longevity genes in both animal models and humans. Human family studies have indicated that a modest amount of the overall variation in adult lifespan (approximately 20–30%) is accounted for by genetic factors. But it is not known if genetic factors become increasingly important for survival at the oldest ages. We study the genetic influence on human lifespan and how it varies with age using the almost extinct cohorts of Danish, Finnish and Swedish twins born between 1870 and 1910 comprising 20,502 individuals followed until 2003–2004. We first estimate mean lifespan of twins by lifespan of co-twin and then turn to the relative recurrence risk of surviving to a given age. Mean lifespan for male monozygotic (MZ) twins increases 0.39 [95% CI (0.28, 0.50)] years for every year his co-twin survives past age 60 years. This rate is significantly greater than the rate of 0.21 (0.11, 0.30) for dizygotic (DZ) males. Females and males have similar rates and these are negligible before age 60 for both MZ and DZ pairs. We moreover find that having a co-twin surviving to old ages substantially and significantly increases the chance of reaching the same old age and this chance is higher for MZ than for DZ twins. The relative recurrence risk of reaching age 92 is 4.8 (2.2, 7.5) for MZ males, which is significantly greater than the 1.8 (0.10, 3.4) for DZ males. The patterns for females and males are very similar, but with a shift of the female pattern with age that corresponds to the better female survival. Similar results arise when considering only those Nordic twins that survived past 75 years of age. The present large population based study shows genetic influence on human lifespan. While the estimated overall strength of genetic influence is compatible with previous studies, we find that genetic influences on lifespan are minimal prior to age 60 but increase thereafter. These findings provide a support for the search for genes affecting longevity in humans, especially at advanced ages.